Alexander W. Wyatt

Androgen receptor gene aberrations in circulating cell-free DNA: biomarkers of therapeutic resistance in castration-resistant prostate cancer

Purpose: Although novel agents targeting the androgen–androgen receptor (AR) axis have altered the treatment paradigm of metastatic castration-resistant prostate cancer (mCRPC), development of therapeutic resistance is inevitable. In this study, we examined whether AR gene aberrations detectable in circulating cell-free DNA (cfDNA) are associated with resistance to abiraterone acetate and enzalutamide in mCRPC patients. Experimental Design: Plasma was collected from 62 mCRPC patients ceasing abiraterone acetate (n = 29), enzalutamide (n = 19), or other agents (n = 14) due to disease progression. DNA was extracted and subjected to array comparative genomic hybridization (aCGH) for chromosome copy number analysis, and Roche 454 targeted next-generation sequencing of exon 8 in the AR. Results: On aCGH, AR amplification was significantly more common in patients progressing on enzalutamide than on abiraterone or other agents (53% vs. 17% vs. 21%, P = 0.02, χ2). Missense AR exon 8 mutations were detected in 11 of 62 patients (18%), including the first reported case of an F876L mutation in an enzalutamide-resistant patient and H874Y and T877A mutations in 7 abiraterone-resistant patients. In patients switched onto enzalutamide after cfDNA collection (n = 39), an AR gene aberration (copy number increase and/or an exon 8 mutation) in pretreatment cfDNA was associated with adverse outcomes, including lower rates of PSA decline ≥ 30% (P = 0.013, χ2) and shorter time to radiographic/clinical progression (P = 0.010, Cox proportional hazards regression). Conclusions: AR gene aberrations in cfDNA are associated with resistance to enzalutamide and abiraterone in mCRPC. Our data illustrate that genomic analysis of cfDNA is a minimally invasive method for interrogating mechanisms of therapeutic resistance in mCRPC. Clin Cancer Res; 21(10); 2315–24. ©2015 AACR.

High Fidelity Patient-Derived Xenografts for Accelerating Prostate Cancer Discovery and Drug Development

Standardized and reproducible preclinical models that recapitulate the dynamics of prostate cancer are urgently needed. We established a bank of transplantable patient-derived prostate cancer xenografts that capture the biologic and molecular heterogeneity currently confounding prognostication and therapy development. Xenografts preserved the histopathology, genome architecture, and global gene expression of donor tumors. Moreover, their aggressiveness matched patient observations, and their response to androgen withdrawal correlated with tumor subtype. The panel includes the first xenografts generated from needle biopsy tissue obtained at diagnosis. This advance was exploited to generate independent xenografts from different sites of a primary site, enabling functional dissection of tumor heterogeneity. Prolonged exposure of adenocarcinoma xenografts to androgen withdrawal led to castration-resistant prostate cancer, including the first-in-field model of complete transdifferentiation into lethal neuroendocrine prostate cancer. Further analysis of this model supports the hypothesis that neuroendocrine prostate cancer can evolve directly from adenocarcinoma via an adaptive response and yielded a set of genes potentially involved in neuroendocrine transdifferentiation. We predict that these next-generation models will be transformative for advancing mechanistic understanding of disease progression, response to therapy, and personalized oncology.

Genomic Alterations in Cell-Free DNA and Enzalutamide Resistance in Castration-Resistant Prostate Cancer

Importance The molecular landscape underpinning response to the androgen receptor (AR) antagonist enzalutamide in patients with metastatic castration-resistant prostate cancer (mCRPC) is undefined. Consequently, there is an urgent need for practical biomarkers to guide therapy selection and elucidate resistance. Although tissue biopsies are impractical to perform routinely in the majority of patients with mCRPC, the analysis of plasma cell-free DNA (cfDNA) has recently emerged as a minimally invasive method to explore tumor characteristics. Objective To reveal genomic characteristics from cfDNA associated with clinical outcomes during enzalutamide treatment. Design, Setting, and Participants Plasma samples were obtained from August 4, 2013, to July 31, 2015, at a single academic institution (British Columbia Cancer Agency) from 65 patients with mCRPC. We collected temporal plasma samples (at baseline, 12 weeks, end of treatment) for circulating cfDNA and performed array comparative genomic hybridization copy number profiling and deep AR gene sequencing. Samples collected at end of treatment were also subjected to targeted sequencing of 19 prostate cancer-associated genes. Exposure Enzalutamide, 160 mg, daily orally. Main Outcomes and Measures Prostate-specific antigen response rate (decline ≥50% from baseline confirmed ≥3 weeks later). Radiographic (as per Prostate Cancer Working Group 2 Criteria) and/or clinical progression (defined as worsening disease-related symptoms necessitating a change in anticancer therapy and/or deterioration in Eastern Cooperative Group performance status ≥2 levels). Results The 65 patients had a median (interquartile range) age of 74 (68-79) years. Prostate-specific antigen response rate to enzalutamide treatment was 38% (25 of 65), while median clinical/radiographic progression-free survival was 3.5 (95% CI, 2.1-5.0) months. Cell-free DNA was isolated from 122 of 125 plasma samples, and targeted sequencing was successful in 119 of 122. AR mutations and/or copy number alterations were robustly detected in 48% (31 of 65) and 60% (18 of 30) of baseline and progression samples, respectively. Detection of AR amplification, heavily mutated AR (≥2 mutations), and RB1 loss were associated with worse progression-free survival, with hazard ratios of 2.92 (95% CI, 1.59-5.37), 3.94 (95% CI, 1.46-10.64), and 4.46 (95% CI, 2.28-8.74), respectively. AR mutations exhibited clonal selection during treatment, including an increase in glucocorticoid-sensitive AR L702H and promiscuous AR T878A in patients with prior abiraterone treatment. At the time of progression, cfDNA sequencing revealed mutations or copy number changes in all patients tested, including clinically actionable alterations in DNA damage repair genes and PI3K pathway genes, and a high frequency (4 of 14) of activating CTNNB1 mutations. Conclusions and Relevance Clinically informative genomic profiling of cfDNA was feasible in nearly all patients with mCRPC and can provide important insights into enzalutamide response and resistance.

The Placental Gene PEG10 Promotes Progression of Neuroendocrine Prostate Cancer

More potent targeting of the androgen receptor (AR) in advanced prostate cancer is driving an increased incidence of neuroendocrine prostate cancer (NEPC), an aggressive and treatment-resistant AR-negative variant. Its molecular pathogenesis remains poorly understood but appears to require TP53 and RB1 aberration. We modeled the development of NEPC from conventional prostatic adenocarcinoma using a patient-derived xenograft and found that the placental gene PEG10 is de-repressed during the adaptive response to AR interference and subsequently highly upregulated in clinical NEPC. We found that the AR and the E2F/RB pathway dynamically regulate distinct post-transcriptional and post-translational isoforms of PEG10 at distinct stages of NEPC development. In vitro, PEG10 promoted cell-cycle progression from G0/G1 in the context of TP53 loss and regulated Snail expression via TGF-β signaling to promote invasion. Taken together, these findings show the mechanistic relevance of RB1 and TP53 loss in NEPC and suggest PEG10 as a NEPC-specific target.

Targeting the adaptive molecular landscape of castration-resistant prostate cancer.

Castration and androgen receptor (AR) pathway inhibitors induce profound and sustained responses in advanced prostate cancer. However, the inevitable recurrence is associated with reactivation of the AR and progression to a more aggressive phenotype termed castration‐resistant prostate cancer (CRPC). AR reactivation can occur directly through genomic modification of the AR gene, or indirectly via co‐factor and co‐chaperone deregulation. This mechanistic heterogeneity is further complicated by the stress‐driven induction of a myriad of overlapping cellular survival pathways. In this review, we describe the heterogeneous and evolvable molecular landscape of CRPC and explore recent successes and failures of therapeutic strategies designed to target AR reactivation and adaptive survival pathways. We also discuss exciting areas of burgeoning anti‐tumour research, and their potential to improve the survival and management of patients with CRPC.

nFuse: Discovery of complex genomic rearrangements in cancer using high-throughput sequencing

Complex genomic rearrangements (CGRs) are emerging as a new feature of cancer genomes. CGRs are characterized by multiple genomic breakpoints and thus have the potential to simultaneously affect multiple genes, fusing some genes and interrupting other genes. Analysis of high-throughput whole-genome shotgun sequencing (WGSS) is beginning to facilitate the discovery and characterization of CGRs, but further development of computational methods is required. We have developed an algorithmic method for identifying CGRs in WGSS data based on shortest alternating paths in breakpoint graphs. Aiming for a method with the highest possible sensitivity, we use breakpoint graphs built from all WGSS data, including sequences with ambiguous genomic origin. Since the majority of cell function is encoded by the transcriptome, we target our search to find CGRs that underlie fusion transcripts predicted from matched high-throughput cDNA sequencing (RNA-seq). We have applied our method, nFuse, to the discovery of CGRs in publicly available data from the well-studied breast cancer cell line HCC1954 and primary prostate tumor sample 963. We first establish the sensitivity and specificity of the nFuse breakpoint prediction and scoring method using breakpoints previously discovered in HCC1954. We then validate five out of six CGRs in HCC1954 and two out of two CGRs in 963. We show examples of gene fusions that would be difficult to discover using methods that do not account for the existence of CGRs, including one important event that was missed in a previous study of the HCC1954 genome. Finally, we illustrate how CGRs may be used to infer the gene expression history of a tumor.

Functional analysis of androgen receptor mutations that confer anti-androgen resistance identified in circulating cell-free DNA from prostate cancer patients

BackgroundThe androgen receptor (AR) is a pivotal drug target for the treatment of prostate cancer, including its lethal castration-resistant (CRPC) form. All current non-steroidal AR antagonists, such as hydroxyflutamide, bicalutamide, and enzalutamide, target the androgen binding site of the receptor, competing with endogenous androgenic steroids. Several AR mutations in this binding site have been associated with poor prognosis and resistance to conventional prostate cancer drugs. In order to develop an effective CRPC therapy, it is crucial to understand the effects of these mutations on the functionality of the AR and its ability to interact with endogenous steroids and conventional AR inhibitors.ResultsWe previously utilized circulating cell-free DNA (cfDNA) sequencing technology to examine the AR gene for the presence of mutations in CRPC patients. By modifying our sequencing and data analysis approaches, we identify four additional single AR mutations and five mutation combinations associated with CRPC. Importantly, we conduct experimental functionalization of all the AR mutations identified by the current and previous cfDNA sequencing to reveal novel gain-of-function scenarios. Finally, we evaluate the effect of a novel class of AR inhibitors targeting the binding function 3 (BF3) site on the activity of CRPC-associated AR mutants.ConclusionsThis work demonstrates the feasibility of a prognostic and/or diagnostic platform combining the direct identification of AR mutants from patients’ serum, and the functional characterization of these mutants in order to provide personalized recommendations regarding the best future therapy.

Integrated genome and transcriptome sequencing identifies a novel form of hybrid and aggressive prostate cancer

Next‐generation sequencing is making sequence‐based molecular pathology and personalized oncology viable. We selected an individual initially diagnosed with conventional but aggressive prostate adenocarcinoma and sequenced the genome and transcriptome from primary and metastatic tissues collected prior to hormone therapy. The histology‐pathology and copy number profiles were remarkably homogeneous, yet it was possible to propose the quadrant of the prostate tumour that likely seeded the metastatic diaspora. Despite a homogeneous cell type, our transcriptome analysis revealed signatures of both luminal and neuroendocrine cell types. Remarkably, the repertoire of expressed but apparently private gene fusions, including C15orf21:MYC, recapitulated this biology. We hypothesize that the amplification and over‐expression of the stem cell gene MSI2 may have contributed to the stable hybrid cellular identity. This hybrid luminal‐neuroendocrine tumour appears to represent a novel and highly aggressive case of prostate cancer with unique biological features and, conceivably, a propensity for rapid progression to castrate‐resistance. Overall, this work highlights the importance of integrated analyses of genome, exome and transcriptome sequences for basic tumour biology, sequence‐based molecular pathology and personalized oncology. Copyright

Generation 2.5 Antisense Oligonucleotides Targeting the Androgen Receptor and Its Splice Variants Suppress Enzalutamide-Resistant Prostate Cancer Cell Growth

Purpose: Enzalutamide (ENZ) is a potent androgen receptor (AR) antagonist with activity in castration-resistant prostate cancer (CRPC); however, progression to ENZ-resistant (ENZ-R) CRPC frequently occurs with rising serum PSA levels, implicating AR full-length (ARFL) or variants (AR-Vs) in disease progression. Experimental Design: To define functional roles of ARFL and AR-Vs in ENZ-R CRPC, we designed 3 antisense oligonucleotides (ASO) targeting exon-1, intron-1, and exon-8 in AR pre-mRNA to knockdown ARFL alone or with AR-Vs, and examined their effects in three CRPC cell lines and patient-derived xenografts. Results: ENZ-R-LNCaP cells express high levels of both ARFL and AR-V7 compared with CRPC-LNCaP; in particular, ARFL levels were approximately 12-fold higher than AR-V7. Both ARFL and AR-V7 are highly expressed in the nuclear fractions of ENZ-R-LNCaP cells even in the absence of exogenous androgens. In ENZ-R-LNCaP cells, knockdown of ARFL alone, or ARFL plus AR-Vs, similarly induced apoptosis, suppressed cell growth and AR-regulated gene expression, and delayed tumor growth in vivo. In 22Rv1 cells that are inherently ENZ-resistant, knockdown of both ARFL and AR-Vs more potently suppressed cell growth, AR transcriptional activity, and AR-regulated gene expression than knockdown of ARFL alone. Exon-1 AR-ASO also inhibited tumor growth of LTL-313BR patient-derived CRPC xenografts. Conclusions: These data identify the AR as an important driver of ENZ resistance, and while the contributions of ARFL and AR-Vs can vary across cell systems, ARFL is the key driver in the ENZ-R LNCaP model. AR targeting strategies against both ARFL and AR-Vs is a rational approach for AR-dependent CRPC. Clin Cancer Res; 21(7); 1675–87. ©2015 AACR.

Bone morphogenetic protein 7 (BMP7) mutations are associated with variable ocular, brain, ear, palate, and skeletal anomalies†

Bone morphogenetic protein (BMP) signaling regulates a range of cellular processes and plays an important role in the specification and patterning of the early embryo. However, due to the functional redundancy of BMP ligands and receptors in tissues where they are coexpressed, relatively little is known about the role of individual BMP ligands in human disease. Here we report heterozygous variations in BMP7, including a frameshift, missense, and Kozak sequence mutation, in individuals with developmental eye anomalies and a range of systemic abnormalities, including developmental delay, deafness, scoliosis, and cleft palate. We determined that BMP7 is expressed in the developing eye, brain, and ear in human embryos in a manner consistent with the phenotype seen in our mutation cases. These data establish BMP7 as an important gene in human eye development, and suggest that BMP7 should be considered during clinical evaluation of individuals with developmental eye anomalies. Hum Mutat 31:1–7, 2010.